Biomedical subjects

Thomas H Foster

Publications and source records attributed to Thomas H Foster.

At least 19 recordsLinked to original sources

PubMed2006

Photodynamic therapy mediates the oxygen-independent activation of hypoxia-inducible factor 1alpha.

Photodynamic therapy (PDT) induces the expression of the hypoxia-inducible factor 1alpha (HIF-1alpha) subunit of the HIF-1 transcription factor and its target genes in vitro and in vivo. PDT also induces the expression of the enzyme cyclooxygenase-2 and its metabolite, prostaglandin E2 (PGE2). PGE2 and hypoxia act independently and synergistically to increase HIF-1alpha accumulation and nuclear translocation. To examine the expression of HIF-1 target genes in response to PDT-mediated oxidative stress and PGE2 under normoxic conditions, we established EMT6 cells transfected with a plasmid consisting of a hypoxia response element promoter and a downstream gene encoding for green fluorescent protein (GFP). To examine the temporal kinetics of HIF-1alpha nuclear translocation in response to PDT, we transfected a second line of EMT6 cells with a GFP-tagged HIF-1alpha fusion vector. Cell monolayers were incubated with 1 microg mL(-1) Photofrin for 24 h and irradiated with fluences of 1, 2.5, and 5 J cm(-2). Direct measurement of oxygen concentration during irradiation confirmed that cells remained well oxygenated. Cells were also exposed to 1 and 10 micromol/L PGE2 for 3 h. In normoxic conditions, Photofrin, PDT, and PGE2 treatment activated HIF-1alpha and induced its nuclear translocation. Maximal Photofrin-PDT-mediated HIF-1alpha activation was intermediate in magnitude between that induced by PGE2 and that by the hypoxia mimic cobalt chloride. This work establishes that PDT induces significant activation of the HIF-1alpha pathway in the absence of hypoxia and supports the interpretation that the induction of HIF-1 target genes by PDT may be mediated, at least in part, by the prostaglandin pathway.

Animals↗

PubMed2006

Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization.

We present an experimental and theoretical study of confocal fluorescence polarization microscopy in turbid media. We have performed an experimental study using a fluorophore-embedded polymer rod immersed in aqueous suspensions of 0.1 and 0.5 microm diameter polystyrene microspheres. A Monte Carlo approach to simulate confocal fluorescence polarization imaging in scattering media is also presented. It incorporates a detailed model of polarized fluorescence generation that includes sampling of elliptical polarization, excited-state molecular rotational Brownian motion, and dipole fluorescence emission. Using both approaches, we determine the effects of the number of scattering events, target depth, photon scattering statistics, objective numerical aperture, and pinhole size on confocal anisotropy imaging. From this detailed analysis and comparison of experiment with simulation, we determine that fluorescence polarization is maintained to depths at which meaningful intensity images can be acquired.

Colloids↗

PubMed2006

Preferential attachment of peritoneal tumor metastases to omental immune aggregates and possible role of a unique vascular microenvironment in metastatic survival and growth.

Controlling metastases remains a critical problem in cancer biology. Within the peritoneal cavity, omental tissue is a common site for metastatic disease arising from intraperitoneal tumors; however, it is unknown why this tissue is so favorable for metastatic tumor growth. Using five different tumor cell lines in three different strains of mice, we found that the omentum was a major site of metastases growth for intraperitoneal tumors. Furthermore, initial attachment and subsequent growth were limited to specific sites within the omentum, consisting of organized aggregates of immune cells. These immune aggregates contained a complex network of capillaries exhibiting a high vascular density, which appear to contribute to the survival of metastatic cells. We found that the vasculature within these aggregates contained CD105+ vessels and vascular sprouts, both indicators of active angiogenesis. A subset of mesothelial cells situated atop the immune aggregates was found to be hypoxic, and a similar proportion was observed to secrete vascular endothelial growth factor-A. These data provide a physiological mechanism by which metastatic tumor cells preferentially grow at sites rich in proangiogenic vessels, apparently stimulated by angiogenic factors produced by mesothelial cells. These sites provide metastatic cells with a microenvironment highly conducive to survival and subsequent growth.

Animals↗

PubMed2005

Mie theory interpretations of light scattering from intact cells.

We present a Mie theory-based analysis of angularly resolved light scattering from intact cells that involves the product of a particle size distribution rho and the scattering cross section sigma. The analysis shows that the ability to size intracellular organelles depends strongly upon the assumed functional form of the particle size distribution; however, the product sigma rho extracts the dominant light scatterers regardless of this choice. We examine goniometer measurements from our own and another laboratory and find that this analysis reconciles seemingly conflicting results and confirms that mitochondrial-sized organelles dominate scattering from intact cells in the angular range 50 degrees - 90 degrees.

Algorithms↗

PubMed2005

Recovery of hemoglobin oxygen saturation and intrinsic fluorescence with a forward-adjoint model.

We present two forward-adjoint models for recovering intrinsic fluorescence spectra and hemoglobin oxygen saturation of turbid samples. The first fits measured diffuse reflectance spectra to obtain the absorption and scattering spectra of the medium, and these are then used to correct distortions imposed on the fluorescence spectrum by absorption and scattering. The second fits only the measured fluorescence spectrum to determine simultaneously the amplitudes of absorption and fluorescence basis spectra and scattering parameters. Both methods are validated with Monte Carlo simulations and experimentally in scattering phantoms containing nicotinamide adenine dinucleotide and human erythrocytes. Preliminary measurements from murine tumors in vivo are presented.

Algorithms↗

PubMed2005

Light scattering from intact cells reports oxidative-stress-induced mitochondrial swelling.

Angularly resolved light scattering measurements were performed on suspensions of EMT6 cells and on mitochondria isolated from rabbit liver. Mie theory analysis of the scattering from intact cells indicated that mitochondrial-sized organelles dominated scattering in the range 5-90 degrees . This interpretation was supported by the analysis of scattering from isolated mitochondria. Intact cells were subjected to oxidative stress by photodynamic insult. After 3 h of incubation in the heme precursor aminolevulinic acid hexylester, EMT6 cells accumulated abundant protoporphyrin IX, an endogenous photosensitizer formed in mitochondria. Irradiation of aminolevulinic acid/protoporphyrin IX-sensitized cells with 10 J cm(-2) of 514 nm light led to pronounced changes in angularly resolved light scattering consistent with mitochondrial swelling. Electron microscopy of similarly treated EMT6 cell monolayers showed significant changes in mitochondrial morphology, which included distension of the outer unit membrane and bloating of the internal mitochondrial compartment. Informed by these electron microscopy results, we implemented a coated sphere model to interpret the scattering from intact cells subjected to oxidative stress. The coated sphere interpretation was compatible with the scattering measurements from these cells, whereas simpler Mie theory models based on homogenous swelling were dramatically unsuccessful. Thus, in this system, angularly resolved light scattering reports oxidative-stress-induced changes in mitochondrial morphology.

Aminolevulinic Acid↗

PubMed2005

Sensitivity of Candida albicans germ tubes and biofilms to photofrin-mediated phototoxicity.

Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated photodynamic action against C. albicans and examined whether mechanisms commonly used by microorganisms to subvert either antimicrobial oxidative defenses or antimicrobial therapy, including biofilm formation, were operative. In buffer and defined medium, germ tubes preloaded with Photofrin retained their photosensitivity for up to 2 hours, indicating the absence of degradation or export of Photofrin by the organism. The addition of serum resulted in a gradual loss of photosensitivity over 2 hours. In contrast to an adaptive response by germ tubes to oxidative stress by hydrogen peroxide, there was no adaptive response to singlet oxygen-mediated stress by photodynamic action. C. albicans biofilms were sensitive to Photofrin-mediated phototoxicity in a dose-dependent manner. Finally, the metabolic activity of C. albicans biofilms following photodynamic insult was significantly lower than that of biofilms treated with amphotericin B for the same time period. These results demonstrate that several of the mechanisms microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated photodynamic treatment of C. albicans. These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, mode of therapy against cutaneous and mucocutaneous candidiasis.

Amphotericin B↗

PubMed2004

Photobleaching kinetics of Photofrin in vivo and in multicell tumour spheroids indicate two simultaneous bleaching mechanisms.

We present a detailed investigation of Photofrin photobleaching and photoproduct accumulation. Fisher rats were sensitized with 10 mg kg(-1) Photofrin and irradiated 24 h later with 514 nm light at 5 or 100 mW cm(-2). Fluorescence spectra were collected from the skin throughout treatment, and sensitizer bleaching and fluorescent photoproduct formation were quantified using spectral analysis. Photofrin bleaching was slightly more rapid at the higher irradiance under these conditions. However, accumulation of photoproduct was significantly enhanced at lower irradiance. To interpret these unexpected findings, we developed a new mathematical model in which reactions between singlet oxygen (1O2) and the photosensitizer and reactions between the sensitizer triplet and biological targets are both allowed to contribute to bleaching. Predictions of this model were tested in experiments performed on EMT6 spheroids sensitized with concentrations of 2.5, 10 and 30 microg mL(-1) Photofrin and subjected to PDT. Photofrin bleaching and photoproduct formation in these spheroids were measured using confocal fluorescence spectroscopy. In qualitative agreement with the mixed-mechanism model predictions, at the highest drug concentration Photofrin bleaching was more efficient via 1O2 reactions, while at the lowest concentration triplet reactions were more efficient. At all concentrations, photoproduct accumulation was greater under conditions of abundant oxygen.

Animals↗

PubMed2004

Carbogen breathing significantly enhances the penetration of red light in murine tumours in vivo.

We report results of experiments that evaluated the influence of oxygenation on the penetration of red light in tissue, with particular emphasis on 630 and 650 nm laser wavelengths commonly used in photodynamic therapy (PDT) of solid tumours. Direct measurements in tissue-simulating phantoms comprised of intact human erythrocytes suspended in a scattering emulsion demonstrated significant enhancements in fluence rate at depths of 0.5-2 cm from the irradiated surface when the cells were fully oxygenated versus fully deoxygenated. The 630 and 650 nm fluence rates at depth in the homogeneous phantoms continued to increase when examined over a continuous range of oxygen partial pressures from 0 to 160 Torr. When considered as a function of haemoglobin oxygen saturation, the largest increases in fluence rate were observed as the saturation increased beyond 70%. Dramatic increases in optical fluence rate were measured at the base of 1-cm-thick subcutaneous EMT6 mammary carcinomas in vivo when the tumour-bearing mouse was subjected to carbogen through a nose cone. These results indicate that improved tumour oxygenation is important in PDT not only for the maintenance of the oxygen-dependent photochemistry but, through the effects reported here, may also enable more efficient treatment of thicker lesions.

Animals↗

PubMed2004

Effect of pigment packaging on diffuse reflectance spectroscopy of samples containing red blood cells.

We present the results of diffuse reflectance measurements made on the surface of a tissue-simulating phantom containing intact human erythrocytes. These measurements indicate that the absorption spectrum of hemoglobin in its natural environment is significantly different from that measured in homogeneous fluid solution, especially in the spectral regions of highest absorption. We show that this difference can be explained by the pigment packaging theory developed by Duysens [Biochim. Biophys. Acta 19, 1 (1956)] and that the adoption of basis spectra that take this effect into account improves the accuracy of fitting diffuse reflectance spectra.

Erythrocytes↗

PubMed2004

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy.

We describe a technique for imaging enzyme activity through steady-state fluorescence anisotropy measurements on a per-pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 microm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy-FL-labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five-fold. Fluorescence lifetime and time-resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy-FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy-containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.

Animals↗

PubMed2004

Hemoglobin oxygen saturations in phantoms and in vivo from measurements of steady-state diffuse reflectance at a single, short source-detector separation.

We present a method for the analysis of steady state diffuse reflectance spectra obtained from vascularized tissue or from tissue simulating phantoms at a single, short source-detector separation. This method uses reasonable assumptions about the structure of the reduced scattering spectrum and basis absorption spectra for oxy- and deoxyhemoglobin, which dominate tissue absorption in the visible region of the spectrum. Using a hybrid P3-diffusion description of light propagation, described originally by Hull and Foster [J. Opt. Soc. Am. A 18, 584-599 (2001)] and suitable for short (approximately 1 mm) source-detector separations and optical properties of tissue at visible wavelengths, we create a forward model of the diffuse reflectance with four free parameters. We demonstrate that this model is able to recover accurately the hemoglobin concentrations and scattering properties from synthetic data generated by Monte Carlo simulation and from reflectance spectra acquired from tissue-simulating phantoms containing intact human erythrocytes. We show also that the method is capable of monitoring carbogen-induced changes in murine tumor oxygenation in vivo. The successful implementation of single, short detector separations enables the measurement of intratumor heterogeneities in hemoglobin oxygen saturation and responses to carbogen using a simple fiber-based probe design.

Algorithms↗

PubMed2004

Susceptibility of Candida species to photodynamic effects of photofrin.

The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of >/=3 microg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.

Candida↗

PubMed2003

Confocal fluorescence spectroscopy and anisotropy imaging system.

We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.

Animals↗

PubMed2003

Activation of heat shock protein 70 promoter with meso-tetrahydroxyphenyl chlorin photodynamic therapy reported by green fluorescent protein in vitro and in vivo.

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT-mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue-exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/ GFP-transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.

Animals↗

PubMed2002

Reverse intersystem crossing in rose bengal. II. Fluence dependence of fluorescence following 532 nm laser excitation.

A fluence-dependent fluorescence technique was used to observe reverse intersystem crossing from a certain higher-lying triplet state of rose bengal populated by a single pulse of 532 nm light. The quantum yield of reverse intersystem crossing from this state was determined to be 0.12+/-0.02 for rose bengal in phosphate-buffered saline. The importance of including molecular rotation effects in the analysis of fluorescence resulting from reverse intersystem crossing is discussed. Differences in the photochemical reactivity of upper triplet states in biological systems have been previously hypothesized to result from photophysical differences, particularly substantial differences in their reverse intersystem crossing yields. In this work this hypothesis is analyzed quantitatively, using numerical models of the population dynamics. These models suggest that reverse intersystem crossing alone cannot adequately explain the differences in biological response.

Fluorescence↗

PubMed2002

In vivo mTHPC photobleaching in normal rat skin exhibits unique irradiance-dependent features.

We report measurements performed on the normal skin of rats in vivo, which provide information on the photobleaching kinetics and mechanisms of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC). Loss of mTHPC fluorescence was monitored using in vivo fluorescence spectroscopy during photodynamic therapy (PDT) performed using 650 nm laser irradiation. The bleaching was evaluated for irradiances of 5, 20 and 50 mW cm(-2). Two distinct phases of mTHPC photobleaching were observed. In the first phase there was no obvious irradiance dependence in the loss of fluorescence vs fluence. The second phase was initiated by an irradiance-dependent discontinuity in the slope of the bleaching curve, after which the photobleaching rates showed an irradiance dependence consistent with an oxygen-dependent reaction process. To investigate the unusual shape of the in vivo bleaching curves, we measured the PDT-induced changes in O2 concentrations in mTHPC-sensitized spheroids irradiated with 2, 5 and 20 mW cm(-2) of 650 nm light. The oxygen concentration data indicated no unusual features within the range of fluences where the discontinuities in fluorescence were observed during in vivo spectroscopy. The fluorescence from the in vivo bleaching experiments thus reports a phenomenon that is not reported by measurements of the photochemical oxygen consumption in the spheroids.

Animals↗

PubMed2002

Effect of irradiation fluence rate on the efficacy of photodynamic therapy and tumor oxygenation in meta-tetra (hydroxyphenyl) chlorin (mTHPC)-sensitized HT29 xenografts in nude mice.

We present direct experimental evidence of the fluence-rate-dependent, radiation-induced variations in intratumor oxygen partial pressure (pO(2)) in HT29 human colon adenocarcinoma xenografts subjected to meta-tetra(hydroxyphenyl)chlorin (mTHPC)-based photodynamic therapy (PDT). The data establish a correlation between tumor oxygenation and treatment outcome. Tumor-bearing mice were injected with 0.3 mg/kg photosensitizer and subjected 72 h later to a 12 J/cm(2) red light dose administered at fluence rates of 5, 30, 90 and 160 mW/cm(2). A significant decrease in mean and median pO(2) was registered at approximately half of the total radiation fluence was delivered in tumors treated at rates of 160 and 90 mW/cm(2). Conversely, with the two lower fluence rates, intratumor pO(2) was maintained at levels comparable to those measured before illumination. Tumor oxygenation values registered shortly after every treatment protocol were at least equal to baseline levels, thus excluding the possibility of significant acute vessel damage during illumination. The tumor regrowth profile correlated with the pO(2) values monitored during irradiation. Tumors treated with fluence rates of 5 and 30 mW/cm(2) exhibited significantly longer tumor quadrupling times than those treated at 160 and 90 mW/cm(2). Improved tumor destruction could be expected by reducing the rate and the extent of oxygen depletion during meta-tetra(hydroxyphenyl)chlorin photodynamic therapy using low fluence rates.

Adenocarcinoma↗